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Comparison of the
optimized conditions for genotyping of ACE ID polymorphism
using conventional and direct blood PCR
Syed Kashif Nawaz and
Shahida Hasnain*
Department
of Microbiology and Molecular Genetics, University of the
Punjab Lahore, Pakistan.
*Corresponding author. E-mail:
genetic@brain.net.pk.
Tel.: +92-42-9231238.
Fax:
+92-42-9230481.
Accepted
31 August, 2009 |
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ACE ID polymorphism is inevitable for genetic epidemiology of
several cardiovascular and non cardiovascular diseases due
to its direct influence on ACE activity level. In the
present work, conditions were optimized for its analysis
using conventional and direct blood PCR (DB PCR). Blood
samples from nine normotensive male donors preserved in EDTA
and lithium-heparin coated vacuatainers separately were used
directly as template for DB PCR. Genomic DNA was isolated
from each vacuatainer for the conventional PCR and DB PCR
also. Conditions were optimized by adjusting the suitable
annealing temperature, amount of MgCl2 (in case
of conventional PCR) and amount of blood used as DNA
template for DB PCR. In case of DNA from EDTA treated blood,
maximum amplification of target sequence occurred at 53oC
with 2 mM concentration of MgCl2 in all samples.
However, when DNA from lithium heparin treated blood was
used as template, 6 out of 9 samples gave amplification
results with 4 mM concentration of MgCl2 at the
same temperature. When 1 µl genomic DNA from EDTA and
lithium heparin treated blood was used as DNA template in DB
PCR, all samples gave maximum yield at 53oC. DB
PCR successfully amplified the target region when 1 µl blood
treated with EDTA and 0.5 µl lithium heparin treated blood
was used per 50 microliter reaction mixture at 51oC
as annealing temperature. It can be concluded from the study
that EDTA treated blood is more suitable for conventional
and DB PCR.
Key
words:
ACE ID polymorphism, step down PCR, direct blood PCR. |
